Smartphone-Based Diagnosis of Parasitic Infections With Colorimetric Assays in Centrifuge Tubes

A smartphone-based platform for the diagnosis of parasitic infections has been developed, tested and validated. The system is capable of making automatic and accurate analysis of millimetric colorimetric arrays in centrifuge collection tubes, which are well established tools used in clinical analysis. To that end, an Android-based software application has been developed, making use of the smartphone rear camera, enabling precise image processing of the colorimetric spot arrays. A low-cost plastic accessory has been developed using 3D-printing to provide controlled illumination, xed sample positioning and cell phone attachment. The platform was then tested repeatedly for its size detection, edge blurriness and colour detection capabilities. A minimum spot radius of 175 m is detectable when using the developed app, with a tolerance of 15%, corresponding to 0.25%of the area where the spot array is printed. Spot edge de nition has been studied up to 40% of blurriness, resulting in a low average percentage error of 1.24%. Colour detection follows the well-known Gamma correction function. Finally, the whole platform was tested and validated using real DNA to analyse for accurate discrimination of Trypanosomatid species, which are responsible for devastating diseases in humans and livestock. The smartphone-based platform can be further extended to other clinical analysis. Its simplicity and reliable performance mean it can be used in remote, limited-resource settings by relatively unskilled technicians/nurses, where diagnostic laboratories are sparsely distributed. The results can however be sent easily via the smartphone to medical experts as well as government health agencies.This work was supported in part by the Spanish Ministry of Economics and Competitivity under Project CTQ2016-78754-C2-1-R, in part by the European Regional Development Fund (ERDF), and in part by the DestiNA Genomica SL provided reagents, samples and Spin-Tube devices. The work of P. Escobedo was supported by the Spanish Ministry of Education, Culture and Sport (MECD), under Grant (FPU13/05032)

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app

Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2023.115268The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (similar to 3 h/test), cost (similar to epsilon 1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento CV20-77741, A-FQM-760-UGR20, PID 2019-110987RB-I00, PID 2019-103938RB-I00Spanish MCIN/AEI P18-RT-2961, P18-TP-4160FEDER/Junta de Andalucia-Consejeria de Salud y Familias PIP-0232-2021European CommissionMCIN/AEI PTQ 2020-011388, IJC 2020-043307-IEuropean Union NextGenerationEU/PRTR PTQ 2020-011388, IJC 2020-043307-

Development and evaluation of robust detection methods for soil transmitted helminths of public health importance in wastewater

Ascaris lumbricoides, a major soil-transmitted helminth infecting over 1.2 billion people worldwide, is most refractory to treatment processes and its presence and/or inactivation in treated material can serve as an indicator of the efficiency of the disinfection process. Due to its high infectivity, risk to children, and resistance to adverse conditions such as high temperatures and alkaline pH, the Ascaris genus are parasites of great significance for evaluation of the sanitization of wastewater and sewage sludge. The reliable quantitative detection of viable ova is therefore essential for both accurate assessment of risk based target values and for the validation of the performance of sanitation systems. Conventional methods of helminth ova enumeration such as microscopy are time-consuming and tedious. The development of polymerase chain reaction (PCR)-based techniques have shown some success in overcoming a number of the challenges associated with conventional techniques, but these techniques also have shortfalls. The overarching aim of this thesis was to develop methods that assure high accuracy, speed and precision in the detection and determination of the viability of Ascaris ova in wastewater. In addition, the importance of accurately estimating the concentration of ova from wastewater and sludge samples was identified. Therefore, a new method to enumerate and recover Ascaris suum ova in wastewater and sludge was developed. The effectiveness of the modified method was compared with the Tulane and double flotation methods. The modified method resulted in enhanced ova recovery from wastewater (> 50%) and sludge (> 60%) with minimal processing time (≤ 2 hours). A significant increase in recovery (P value < 0.0001, paired T-Test) was observed when used to recover ova from wastewater and sludge. PCR-based techniques, especially quantitative PCR can be used to detect Ascaris ova in wastewater matrices with improved sensitivity and precision. However PCR/qPCR methods are unable to differentiate between viable and non-viable ova and might lead to overestimation of infection intensity. To overcome such limitations, a method was developed using DNA intercalating dye propidium monoazide (PMA) with qPCR for the selective detection of viable Ascaris ova from wastewater. The validated PMA-qPCR method was subsequently compared with existing culture-based and BacLight Live/Dead staining methods for viability determination. The percentage of viability determined were; culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%), respectively. No significant statistical difference (P > 0.005, Fisher Exact Test) among the three methods was determined; however PMA-qPCR based viability determination is preferable due to its speed and accuracy. Thus PMA-qPCR could be a useful tool in the screening of Ascaris infection after mass anthelminthics administration and water sanitation and hygiene implemented countries.    Due to the complexity of the assays and requirement for specialised and sophisticated devices, PCR-based techniques can not readily be adapted to be a point-of-care detection assay or used in resource-limited settings where the disease is mostly endemic. To meet the need for rapid on-site assay in resource-limited settings, recombinase polymerase amplification (RPA) assay coupled with eco-friendly lateral flow (LF) strips was developed and validated for the detection of Ascaris ova in wastewater. The Ascaris RPA-LF assay was able to detect Ascaris in less than 30 minutes with an optimal temperature at 37 ¿C and was more sensitive than PCR-based approaches, with detection at DNA concentrations as low as 2 femtograms. Furthermore, ova from two different helminth genera, Ascaris suum and Trichuris suis were able to be detected as a multiplex RPA-LF assay which could significantly reduce the time and cost of helminth identification. The RPA-LF assay was a sensitive, specific, user-friendly and cost-effective technique for the rapid detection of helminth ova with significant potential as an on-site molecular detection tool. One of the main hindrances for the effective use of molecular methods in the detection of STH ova is the extraction of genomic DNA of good quantity and quality which is due to the presence of the tough outer shell of helminth ova. Commercial DNA extraction kits are generally optimised for the extraction of bacterial genomes; however the ova shell is much tougher than the cell walls of bacteria resulting in a lower DNA yield which affects the sensitivity of subsequent molecular assays. Hence, a simple colorimetry-based assay using the surface property of Ascaris ova for in-situ biosynthesis of gold nanoparticles via reduction of tetrachloroauric acid was developed as an alternative to molecular techniques. Visualisation of a colour change from light yellow to ruby red indicated the synthesis of gold nanoparticles on the surface of Ascaris ova; however no such colour change was observed in Trichuris ova. Scanning electron microscopy (SEM) revealed the outer surface of A, suum ova covered with nanoparticles while elemental analysis using energy dispersive X-ray spectroscopy (EDX) confirmed that the presence of 91 w/w % gold in the ova surface.  Although preliminary experiments were successful in terms of differentiating helminth ova, further validations with wastewater samples are required. The aforementioned studies to quantitatively detect Ascaris ova in wastewater represent significant innovations. Although all methods require further assessment, the research carried out in this thesis has led to significant findings in the diagnostic capabilities for STH, especially Ascaris ova, the neglected pathogen. Consequently, this thesis will support the water companies in Australia via accurate detection and screening of Ascaris in wastewater matrices thus allowing safe reuse of wastewater

Applying loop-mediated isothermal amplification (LAMP) in the diagnosis of malaria, leishmaniasis and trypanosomiasis as point-of-care tests (POCTs)

One of the main objectives of the WHO is controlling transmission of parasitic protozoa vector- borne diseases. A quick and precise diagnosis is critical in selecting the optimal therapeutic regime that avoids unnecessary treatments and the emergence of resistance. Molecular assays based on Loop- Mediated Isothermal Amplification (LAMP) techniques are a good alternative to light microscopy and antigen-based rapid diagnostic tests in developing countries, since they allow for a large amount of genetic material generated from a few copies of DNA, and use primers that lead to high sensitivity and specificity, while the amplification process can be performed in isothermal conditions without the need of sophisticated equipment to interpret the results. In this review, the main advances in the development of LAMP assays for the diagnosis of malaria, leishmaniasis and Chagas' disease are discussed as well as the feasibility of their implementation in developing countries and use as point- of-care diagnostic tests

QRsens:dual-purpose quick response code with built-in colorimetric sensors

QRsens represents a family of Quick Response (QR) sensing codes for in-situ air analysis with a customized smartphone application to simultaneously read the QR code and the colorimetric sensors. Five colorimetric sensors (temperature, relative humidity (RH), and three gas sensors (CO₂, NH₃ and H₂S)) were designed with the aim of proposing two end-use applications for ambient analysis, i.e., enclosed spaces monitoring, and smart packaging. Both QR code and colorimetric sensing inks were deposited by standard screen printing on white paper. To ensure minimal ambient light dependence of QRsens during the real-time analysis, the smartphone application was programmed for an effective colour correction procedure based on black and white references for three standard illumination temperatures (3000, 4000 and 5000 K). Depending on the type of sensor being analysed, this integration achieved a reduction of ∼71 – 87% of QRsens's dependence on the light temperature. After the illumination colour correction, colorimetric gas sensors exhibited a detection range of 0.7–4.1%, 0.7–7.5 ppm, and 0.13–0.7 ppm for CO2, NH3 and H2S, respectively. In summary, the study presents an affordable built-in multi-sensing platform in the form of QRsens for in-situ monitoring with potential in different types of ambient air analysis applications

QRsens: Dual-purpose quick response code with built-in colorimetric sensors

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.snb.2022.133001.QRsens represents a family of Quick Response (QR) sensing codes for in-situ air analysis with a customized smartphone application to simultaneously read the QR code and the colorimetric sensors. Five colorimetric sensors (temperature, relative humidity (RH), and three gas sensors (CO2, NH3 and H2S)) were designed with the aim of proposing two end-use applications for ambient analysis, i.e., enclosed spaces monitoring, and smart packaging. Both QR code and colorimetric sensing inks were deposited by standard screen printing on white paper. To ensure minimal ambient light dependence of QRsens during the real-time analysis, the smartphone application was programmed for an effective colour correction procedure based on black and white references for three standard illumination temperatures (3000, 4000 and 5000 K). Depending on the type of sensor being analysed, this integration achieved a reduction of ~71 – 87% of QRsens’s dependence on the light temperature. After the illumination colour correction, colorimetric gas sensors exhibited a detection range of 0.7–4.1%, 0.7–7.5 ppm, and 0.13–0.7 ppm for CO2, NH3 and H2S, respectively. In summary, the study presents an affordable built-in multi-sensing platform in the form of QRsens for in-situ monitoring with potential in different types of ambient air analysis applications.Spanish MCIN/AEI/10.13039/ 501100011033/ (Projects PID2019–103938RB-I00, ECQ2018–004937- P and grant IJC2020–043307-I)Junta de Andalucía (Projects B- FQM-243-UGR18, P18-RT-2961)European Regional Development Funds (ERDF)European Union NextGenerationEU/PRT

Microscale biosensors for HIV detection and viral load determination

The HIV/AIDS pandemic has killed 39 million people worldwide, and nearly as many people are living with HIV infection today. The global response to this disease has come a long way since the emergence of HIV in the early 1980s, including more than 64 billion USD in international spending between 2002 and 2013 alone [1]. Due to the worldwide effort, HIV infection has been transformed from a death sentence into a manageable, chronic illness that can have limited impact on lifespan when treated properly [2]. Antiretroviral therapy, public health campaigns, and other education and prevention efforts have facilitated an age in which no one, regardless of age, gender, sexual orientation, nationality, or socioeconomic status should face despair on account of this infection. However, barriers persist to bringing proper care to millions of people worldwide, including access to testing and the diagnostic tools necessary for proper administration of therapy. Following serological testing to establish HIV-positive status, the current standard of care requires monitoring of CD4+ T lymphocyte counts and plasma HIV viral load to guide administration of antiretroviral therapy. For many individuals living with HIV worldwide, the expensive and sophisticated laboratory instruments necessary for these measurements are extremely difficult to access due to poor healthcare infrastructure and lack of technical personnel. For those who are capable of bearing the expense and inconvenience of traveling to facilities that can provide one or both of these measurements, continuing care can be hindered by difficulties in patient follow-up. A point-of-care technology capable of performing these essential measurements to HIV therapy, therefore, is a critical need worldwide. Here we explore solutions rooted in micro- and nanotechnology principles to address this immense challenge in global health. Point-of-care diagnostics which meet the following criteria could improve the way that HIV/AIDS is treated, particularly in remote and resource-limited settings: low-cost assays (approximately $10 or less), small sample volumes (approximately 10 μL or less), rapid measurements (approximately 10 minutes or less), as well as technologies that are easy to use and portable. Our expertise in this area began with the development of a lab-on-a-chip micro-cytometer for CD4+ T lymphocyte enumeration from a drop of whole blood, which was tested on HIV-positive patients in the Champaign-Urbana, IL area and matched results from clinical flow cytometry at Carle Foundation Hospital in Urbana, IL [3]. This thesis describes work on the complementary measurement, viral load detection, aimed at meeting the ideal criteria described above for a point-of-care diagnostic technology. Our approaches to viral load measurements follow two broad themes. First, we describe an antigen-based approach which leverages immuno-affinity recognition for whole virus particle detection. In this method, the novel component of our sensing system is an ion-filled liposome which, upon stimulation (in this case, by heating), releases ions into low-conductivity media in a microchannel and can be quantitatively measured by a simple impedance measurement. We have termed this technique “ion-release impedance spectroscopy.” Employing the liposome in an immunoassay involving a primary capture antibody to HIV surface proteins and a secondary, identical antibody anchored to the exterior of the liposome, we are able to show qualitative detection of HIV virions in a microchannel [4]. We have improved aspects of this approach by performing ion-release impedance spectroscopy with liposomes exhibiting a higher melting temperature, and explored immuno-affinity capture of viruses on magnetic beads in an attempt to perform a concentration or separation step from a whole blood sample. Our second approach is detection of viral RNA in whole blood. In this technique, we employ loop-mediated isothermal amplification (LAMP) in the detection of viral RNA following a reverse-transcription test. One novel aspect of this approach is in performing the test from unprocessed whole blood, which we introduce into a microfluidic channel, mix with cell lysis buffer, add to RT-LAMP reagents, and distribute into nanoliter-scale droplets on a silicon microchip. Another novel step is to image this reaction with a consumer mobile smartphone device, which we integrate with the microchip setup using a 3-D printed platform. Results from our smartphone-imaged RT-LAMP technique show amplification in reactions containing as few as 3 virus particles per droplet, corresponding to 670 viruses per microliter of whole blood [5]. The true power of this approach, however, can be realized in a quantitative digital detection approach for which we describe a framework and preliminary designs, providing a basis for a highly-practical viral load test based on the proof-of-concept demonstrated in our lab. These micro- and nanotechnology approaches to HIV viral load measurements give hope for a portable diagnostics platform which could bring the standard of life-saving HIV/AIDS care to people in all parts of the world, no matter how remote or resource-limited

Printed Flexible Temperature Sensor with NFC Interface

Integration of sensors with antennas is becoming popular for compact high-performance wireless sensing systems. In this direction, here we present a silver electrodes and Poly(3,4-ethylenedioxythiophene:polystyrene (PEDOT:PSS) based printed temperature sensor on a flexible PVC substrate. The temperature sensor was characterised using a digital multimeter for a temperature range from 25¤C to 90□C. The sensor showed a 70% change in resistance for the tested temperature range. Further, the sensing part was integrated with a Near Field Communication (NFC) tag with the data obtained semi-quantitatively by means of the intensity of an Light Emittign Diode (LED) connected with the antenna system. In this case, the antenna works as an energy harvester to power an LED indicator connected in series to the resistive temperature sensor. The intensity of the LED, which varies with the increase of temperature, was measured using a lux-meter mobile application. The intensity at 70□C was ~42 lux whereas it decreased down to ~14 lux at room temperature (~25□C). The presented system showed potential use as a smart label in applications requiring temperature monitoring

Flexible Potentiostat Readout Circuit Patch for Electrochemical and Biosensor Applications

This paper presents a miniaturized potentiostat readout circuit patch developed for electrochemical or biosensors. The presented patch has been fabricated on a flexible polyimide substrate using off-the-shelf electronics. In contrast to the traditional bulky equipment for electrochemical analysis, the presented patch is conformable and portable. As a proof of concept, the system has been used for pH measurements in buffer solution (6- 8 pH values) with a printed thick film potentiometric pH sensor having platinum counter electrode. The obtained results are in line with a commercially available potentiostat that has been used for benchmarking